Growing marine fish in fresh water

ABSTRACT

The invention relates to methods, compositions and kits for raising marine fish in freshwater. The methods involve adding at least one Polyvalent Cation Sensing Receptor (PVCR) modulator to the freshwater in an amount sufficient to increase expression and/or sensitivity of at least one PVCR; and adding feed for fish consumption to the freshwater, wherein the feed comprises an amount of NaCl sufficient to contribute to a significant increased level of the PVCR modulator in serum of the marine fish.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/411,076, filed Apr. 9,2003, U.S. Pat. No. 6,854,422, which is a continuation of InternationalApplication No. PCT/US01/31625, which designated the United States andwas filed Oct. 11, 2001, published in English, which is acontinuation-in-part of application Ser. No. 09/687,373, filed Oct. 12,2000, now issued U.S. Pat. No. 6,463,882. The entire teachings of theabove application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Growing marine fish has been generally limited to costal regions orseawater tanks. However, many freshwater aquifers exist, for example, inthe Midwest as potential environments for the raising of marine fish.Until now, attempts to grow marine fish in freshwater have beenunsuccessful.

Growing marine fish in freshwater would provide an opportunity fornon-costal areas to raise marine fish. The ability to grow marine fishin freshwater can provide fresh fish and economic growth to these areas.

Hence, a need exists to determine whether it is possible to adapt amarine fish to freshwater, and if so, understand the biologicalmechanisms that allow a marine fish to do so. In particular, a needexists to grow marine fish in freshwater.

SUMMARY OF THE INVENTION

The present invention relates to methods of growing marine fish infreshwater by increasing or maintaining expression of a receptor,referred to as the Polyvalent Cation Sensing Receptor (PVCR). Theexpression and/or sensitivity of the PVCR is modulated or maintained bysubjecting the marine fish to at least one modulator of the PVCR. Themarine fish are subjected to the modulator when it is added to thefreshwater environment, and optionally, to the feed.

In one embodiment, the present invention is directed toward a method ofgrowing marine fish in freshwater comprising adding at least onePolyvalent Cation Sensing Receptor (PVCR) modulator to freshwater in anamount sufficient to modulate or maintain expression and/or sensitivityof at least one PVCR in one or more tissues; transferring the marinefish to the freshwater and adding feed for fish consumption to themodified freshwater, wherein the feed contains an amount of NaClsufficient to contribute to modulate or maintain levels of said PVCRmodulator in serum of the marine fish. PVCR modulators useful in thepresent invention include a divalent cation, a trivalent cation, anaminoglycoside, an organic polycation, an amino acid, a Type ICalcimimetic, a Type II Calcimimetic, 1,25 dihydroxyvitamin D, acytokine, and macrophage chemotatic peptide-1. The feed suitable in themethods of the present invention contains at least about 1% NaCl byweight and can optionally include a PVCR modulator.

The present invention also encompasses a method of transferring marinefish to freshwater comprising adding at least one Polyvalent CationSensing Receptor (PVCR) modulator to the freshwater in an amountsufficient to modulate or maintain expression and/or sensitivity of atleast one PVCR in one or more tissues, transferring the marine fish tothe freshwater, adding feed for fish consumption to the modifiedfreshwater, wherein the feed contains at least about 1% NaCl by weight.The PVCR modulator can be a PVCR agonist, a divalent cation, a trivalentcation, an aminoglycoside, an organic polycation or an amino acid.

In another embodiment, the present invention is directed toward a methodof growing marine fish in freshwater comprising determining the level ofat least one PVCR modulator in freshwater, adding said PVCR modulator tothe freshwater in an amount sufficient to modulate or maintainexpression and/or sensitivity of at least one PVCR in one or moretissues, transferring the marine fish to the freshwater and adding feedfor fish consumption to the modified freshwater, wherein the feedcontains an amount of NaCl sufficient to modulate or maintain levels ofsaid PVCR modulator in serum of the marine fish (calcium and magnesium).PVCR modulator that can be assessed. The PVCR modulator is added tofreshwater such that the freshwater has between about 0.3 mM and about10.0 mM calcium and between about 0.5 mM and about 10.0 mM magnesiumprior to transferring marine fish.

The present invention is also directed to a method of growing marinefish in freshwater having between about 0.3 mM and about 10.0 mM ofcalcium and between about 0.5 mM and 10.0 mM of magnesium. The methodcomprises adding feed to the freshwater wherein the feed contains anamount of NaCl sufficient to modulate or maintain levels of said PVCRmodulator in serum of the marine fish, wherein modulated or maintainedexpression of at least one PVCR is modulated or maintained in one ormore tissues.

In another embodiment, the present invention is directed toward a methodof transferring marine fish to freshwater comprising transferring themarine fish to freshwater having magnesium and calcium in the freshwaterin amounts sufficient to modulate or maintain the expression and/orsensitivity of at least one PVCR in one or more tissues and adding feedto the freshwater, wherein the feed contains at least about 1% NaCl byweight.

The present invention is also directed to a method of growing flounderin freshwater comprising transferring flounder to freshwater having atleast one PVCR modulator in an amount sufficient to increase or maintainexpression and/or sensitivity of at least one PVCR in one or more tissueand adding feed for fish consumption to the freshwater, wherein the feedcontains an amount of NaCl sufficient to contribute to a significantincreased level of said PVCR modulator in serum of the flounder. The pHof the freshwater should be greater than 7.0.

In another embodiment, the present invention is directed toward anaquatic mixture for providing an environment to transfer marine fish tofreshwater, comprising at least one PVCR modulator. An aquatic mixtureis a medium suitable for transfer of marine fish to freshwater duringaquaculture.

The present invention is also directed to a kit for growing marine fishin freshwater comprising an aquatic mixture for providing an environmentto grow the marine fish, wherein the aquatic mixture comprises at leastone PVCR modulator; and an aquatic food composition containing aconcentration of NaCl between about 10,000 mg/kg and about 100,000mg/kg.

Surprisingly, it has been discovered that modulated or maintainedexpression and/or altering the sensitivity of the PVCR allows thesemarine fish to live and thrive in freshwater. Until the discovery of thepresent invention, the aquaculture industry was unable to transfer themarine fish to freshwater without subjecting the fish to stress, deathand/or disease. Unlike this practice, carrying out the steps of theinvention modulates or maintains the expression and/or alters thesensitivity of the PVCR and allows for transfer of the marine fish tofreshwater with minimal or no stress, death and/or disease, andunexpectedly, the fish grow. In fact, marine fish that grow infreshwater have a higher fat content, and a milder, less “fishy” taste.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are diagrams illustrating the partial nucleotide (SEQ IDNO:1) and amino acid (SEQ ID NO:2) sequences of the PVCR of Cod.

FIGS. 2A and 2B are diagrams illustrating the partial nucleotide (SEQ IDNO:3) and amino acid (SEQ ID NO:4) sequences of the PVCR of Haddock.

FIGS. 3A and 3B are diagrams illustrating the partial nucleotide (SEQ IDNO:5) and amino acid (SEQ ID NO:6) sequences of the PVCR of Hake.

FIGS. 4A-B are diagrams illustrating the partial nucleotide (SEQ IDNO:7) and amino acid (SEQ ID NO:8) sequences of the PVCR of Halibut.

FIG. 5A-B are diagrams illustrating the partial nucleotide (SEQ ID NO:9)and amino acid (SEQ ID NO:10) sequences of the PVCR of Mackerel.

FIGS. 6A-B are diagrams illustrating the partial nucleotide (SEQ IDNO:11) and amino acid (SEQ ID NO:12) sequences of the PVCR of Pollack.

FIG. 7A-B are diagrams illustrating the partial nucleotide (SEQ IDNO:13) and amino acid (SEQ ID NO:14) sequences of the PVCR of Sea Bass.

FIGS. 8A-B are diagrams illustrating the partial nucleotide (SEQ IDNO:15) and amino acid (SEQ ID NO:16) sequences of the PVCR of Swordfish.

FIGS. 9A-B are diagrams illustrating the partial nucleotide (SEQ IDNO:17) and amino acid (SEQ ID NO:18) sequences of the PVCR of Tuna.

FIGS. 10A-C are diagrams illustrating the partial nucleotide (SEQ IDNO:19) and amino acid (SEQ ID NO:20) sequences of the PVCR of WinterFlounder.

FIG. 11 is a diagram illustrating the partial nucleotide (SEQ ID NO: 21)and amino acid (SEQ ID NO: 22) sequences of PVCR of Summer Flounder.

FIGS. 12A-D are diagrams illustrating the alignment of the nucleic acidssequences for Cod (SEQ ID NO: 1), Haddock (SEQ ID NO: 3), Hake (SEQ IDNO: 5), Halibut (SEQ ID NO: 7), Mackerel (SEQ ID NO: 9), Pollock (SEQ IDNO: 11), Sea Bass (SEQ ID NO: 13), Swordfish (SEQ ID NO: 15), Tuna (SEQID NO: 17), Winter Flounder (SEQ ID NO: 19).

FIGS. 13A-C are diagrams illustrating the alignment of the amino acidssequences for Cod (SEQ ID NO: 2), Haddock (SEQ ID NO: 4), Hake (SEQ IDNO: 6), Halibut (SEQ ID NO: 8), Mackerel (SEQ ID NO: 10), Pollock (SEQID NO: 12), Sea Bass (SEQ ID NO: 14), Swordfish (SEQ ID NO: 16), Tuna(SEQ ID NO: 18), Winter Flounder (SEQ ID NO: 20).

FIGS. 14A-B are diagrams illustrating the nucleic acid sequence of SKCaR(SEQ ID NO.: 23).

FIG. 15 is a graphical representation illustrating the growth of summerflounder in freshwater that underwent APS Process I and grown infreshwater for a total of 51 days. Samples of body characteristics offlounders were obtained at (1) prior to placement in freshwater; (2) 20days after placement in freshwater; (3) 30 days after placement infreshwater; and (4) 51 days after placement in freshwater. APS Process Iis defined in Example 2.

FIG. 16 is a graphical representation illustrating the growth of summerflounder in seawater for a total of 51 days. Samples of bodycharacteristics of flounders were obtained at (1) prior to placement inseawater; (2) 20 days after placement in seawater; (3) 30 days afterplacement in seawater; and (4) 51 days after placement in seawater.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for growing or raising marinefish in freshwater. The methods involve modulating or maintainingexpression and/or altering the sensitivity of a Polyvalent CationSensing Receptor (PVCR) (e.g., at least one PVCR). The invention relatesto modulating or maintaining expression of the PVCR that affects thefish's ability to adapt to freshwater.

In particular, the methods of the present invention include adding atleast one PVCR modulator to the freshwater, and adding a specially madeor modified feed to the freshwater for consumption by the fish. The feedcontains a sufficient amount of sodium chloride (NaCl) (e.g., betweenabout 1% and about 10% by weight, or about 10,000 mg/kg to about 100,000mg/kg) to modulate or maintain levels of the PVCR modulator in theserum. This amount of NaCl in the feed causes or induces the marine fishto drink more freshwater. Since the freshwater contains a PVCR modulatorand the fish ingest increased amounts of it, the serum level of the PVCRmodulator significantly increases in the fish, and causes increased ormaintained PVCR expression and/or altered PVCR sensitivity. A“significant” increase is used herein to refer to a measurable rise inthe level or quantity of PVCR or RVCR modulator as compared to a controlor reference. Methods of measuring or detecting a significant increasein PVCR or PVCR modulator are disclosed herein and known to one skilledin the art.

The methods of the present invention pertain to adapting marine fish tofreshwater. Marine fish are fish that live, at least for most of theiradult lives, in seawater. Marine fish include, for example, Cod,Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass, Swordfish, Tuna,Winter Flounder, and Summer Flounder. The marine fish are adapted tofreshwater having a PVCR modulator.

The term “marine fish” is understood by one of skill in the art. Theterm, “freshwater,” means water that comes from, for example, a stream,river, ponds, public water supply, or from other non-marine sourceshaving, for example, the following ionic composition: less than about 2mM of magnesium, calcium and NaCl. The phrases “modified freshwater,”“freshwater as modified by the addition of a PVCR” and “PVCR modulatorenvironment” refer to freshwater to which at least one PVCR modulatorhas been added, as described herein.

The PVCR modulator is added to the freshwater in sufficient amounts tomodulate or maintain expression or alter the sensitivity of at least onePVCR. A PVCR has been isolated from various tissue of several types ofmarine fish using molecular biological techniques. For example, nucleicacid was isolated from tissue samples from various species of marinefish including Cod, Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass,Swordfish, Tuna, Winter Flounder and Summer Flounder. The nucleic acidwas amplified using Polymerase Chain Reaction (PCR) methodology. Theamplified DNA was purified, subcloned into vectors, and their sequenceswere determined, as described in Example 4.

The PVCR, which is located in various tissues (e.g., gill, skin,intestine, kidney, urinary bladder, brain or muscle) of the marine fish,senses alterations in PVCR modulators including various ions (e.g.,divalent cations), for example, in the surrounding water, in their serumor in the luminal contents of tubules inside the body, such as kidney,urinary bladder, or intestine. The ability to sense PVCR modulatorsresults in a modulation or a maintenance in the expression of PVCR,thereby allowing the fish to better adapt to freshwater. Modulated ormaintained expression of the PVCR can occur, for example, in one or moretissues. As used herein, the “sensitivity” of the PVCR refers toalteration of PVCR expression in response to a change in theconcentration of PVCR modulators. PVCR expression can be assessed bymeasuring or detecting PVCR polypeptide or nucleic acid molecules in asample by standard methods.

A “PVCR modulator” is defined herein to mean a compound which modulatesexpression of the PVCR, or modulates the sensitivity or responsivenessof the PVCR, or maintains an already increased PVCR expression level inone or more tissues. Such compounds include, but are not limited to,PVCR agonists (e.g., inorganic polycations, organic polycations andamino acids), Type II calcimimetics, and compounds that indirectly alterPVCR expression (e.g., 1,25 dihydroxyvitamin D in concentrations ofabout 3,000-10,000 International Units/kg feed), cytokines such asInterleukin Beta, and Macrophage Chemotatic Peptide-1 (MCP-1)). Examplesof Type II calcimimetics, which modulate expression and/or sensitivityof the PVCR, are, for example, NPS—R-467 and NPS—R-568 from NPSPharmaceutical Inc., (Salt Lake, Utah, U.S. Pat. Nos. 5,962,314;5,763,569; 5,858,684; 5,981,599; 6,001,884) which can be administered inconcentrations of between about 0.1 μM and about 100 μM feed or water.See Nemeth, E. F. et al., PNAS 95: 4040-4045 (1998).

Examples of inorganic polycations are divalent cations including calciumat a concentration between about 0.3 and about 10.0 mM and magnesium ata concentration between about 0.5 and about 10.0 mM; and trivalentcations including, but not limited to, gadolinium (Gd3+) at aconcentration between about 1 and about 500 μM.

Organic polycations include, but are not limited to, aminoglycosidessuch as neomycin or gentamicin in concentrations of between about 1 andabout 8 gm/kg feed as well as organic polycations including polyamines(e.g., polyarginine, polylysine, polyhistidine, polyornithine, spermine,cadaverine, putricine, copolymers of poly arginine/histidine, polylysine/arginine in concentrations of between about 10 μM and 10 mMfeed). See Brown, E. M. et al., Endocrinology 128: 3047-3054 (1991);Quinn, S. J. et al., Am. J. Physiol. 273: C1315-1323 (1997).

Additionally, PVCR agonists include amino acids such as L-Tryptophan,L-Tyrosine, L-Phenylalanine, L-Alanine, L-Serine, L-Arginine,L-Histidine, L-Leucine, L-Isoleucine, and L-Cystine at concentrations ofbetween about 1 and about 10 gm/kg feed. See Conigrave, A. D., et al.,PNAS 97: 4814-4819 (2000). The molar concentrations refer to free orionized concentrations of the PVCR modulator in the freshwater, and donot include amounts of bound PVCR modulator (e.g., PVCR modulator boundto negatively charged particles including glass, proteins, or plasticsurfaces). Any combination of these modulators can be added to the wateror to the feed (in addition to the NaCl, as described herein), so longas the combination modulates or maintains expression and/or sensitivityof at least one PVCR.

The PVCR modulator can be administered to the fish in a number of ways.The invention encompasses administration of the PVCR in any way that issufficient to modulate or maintain the expression and/or alter thesensitivity of the PVCR. In one embodiment, the PVCR modulator is simplyadded to the freshwater, as described herein. PVCR modulators that areadded to the water increase or maintain or decrease expression and/oralter the sensitivity of the PVCR on the skin and gills of the fish, andcan be ingested by the fish, in particular, when fish are fed feedhaving between about 1% and about 10% NaCl (e.g., in concentrationsbetween about 1 and about 10 gm/100 gm feed). In addition to adding NaClto the feed, the PVCR modulator can also be added to the feed. Amountsand types of PVCR modulators added to the feed are also describedherein. Other embodiments include subjecting the fish to the PVCRmodulator by “dipping” the fish in the modulator, e.g., organicpolycations. The organic polycations can be formulated in such a way asto allow the polycations to adhere to the skin and gills of the fish, insufficient amounts to increase or maintain expression of the PVCR.

The invention also embodies assessing the amounts of existing PVCRmodulator in the freshwater environment and in the serum of fish. PVCRmodulators are assessed or measured using methods known in the art.After assessment, the PVCR modulator is added to the water to bring theconcentration up to an amount sufficient to modulate or maintainexpression and/or sensitivity of at least one PVCR, or sufficient tobring the concentrations of the PVCR modulator within the stated ranges.For example, an aquifer assessed at having only 0.2 mM of calcium needsadditional calcium to bring the concentration up to between about 0.3 mMand between about 10.0 mM.

In a preferred embodiment, the present invention is practiced by addinga combination of two PVCR agonists to the freshwater. In particular,calcium and magnesium are added to the freshwater to bring theconcentrations of each to between about 0.3 mM and about 10.0 mM ofcalcium, and between about 0.5 mM and about 10.0 mM of magnesium. Inaddition to adding calcium and magnesium to the water, these ranges ofion concentrations can be achieved by providing a brackish water (e.g.,diluted seawater) environment for the fish.

Calcium and magnesium can come from a variety of sources, that whenadded to the water, the calcium and/or magnesium levels modulate ormaintain expression of the PVCR, and/or are within the stated ranges.Sources of calcium and magnesium can be a mixture of a variety ofcompounds, or each can come from a substantially uniform or purecompound. Sources of calcium include, for example, Ca(CO₃)₂, CaCl₂, andCaSO₄ and sources of magnesium include, for example, MgCl₂, MgSO₄,MgBr₂, and MgCO₃.

In one embodiment, the invention includes intermittent (e.g.,interrupted) as well as continuous (e.g., non-interrupted) exposure tofreshwater having at least one PVCR modulator, while on the NaCl diet.Intermittent exposure to the PVCR can occur so long as the PVCRexpression and/or altered sensitivity remains modulated or maintained.Continuous maintenance in or exposure to freshwater having at least onePVCR modulator is shown in Example 2.

The marine fish are transferred from seawater. The term, “seawater,”means water that comes from the sea, or water which has been formulatedto simulate the chemical and mineral composition of water from the sea.The major elemental composition of the prepared seawater preferablyfalls substantially within the range of the major elemental compositionof the natural seawater (e.g., having the following ionic composition:greater than 30 mM of magnesium, greater than about 6 mM of calcium, andgreater than about 300 mM NaCl). Methods of preparing artificialseawater are known in the art and are described in, for instance, U.S.Pat. No. 5,351,651.

In one embodiment, the marine fish are treated by the methods of thepresent invention by subjecting the fish to a gradual or step-wisedecrease in salinity for a period of time prior to transfer tofreshwater, while being fed a NaCl diet. Salinity refers to the ionicconcentrations (e.g., calcium, magnesium and sodium) in water. The fishare maintained in a decreasing salinity environment for a sufficientperiod of time to modulate or maintain expression and/or sensitivity ofat least one PVCR. Factors that can influence the length of time tomaintain the fish in a decreased salinity prior to transfer tofreshwater include, but are not limited to, size of the fish, level ofPVCR expression or sensitivity, if any, prior to addition of the PVCRmodulator to the freshwater, the fish's ability to excrete the PVCRmodulator and ions, and the fish's surface to volume ratio. Therefore,the length of time the fish is maintained can range between about 5 daysand about 60 days, and preferably, between about 10 days and about 25days.

The ionic concentrations of seawater are decreased by between about 10%and about 90%, and preferably, between about 25% and about 50%.Combinations of decreasing salinity and various lengths of exposure tothe salinity are encompassed by the invention. For example, as describedin Example 2, fish were adapted to 50% seawater (50% salinity ofseawater) for 10 days, and then adapted to 25% seawater (25% salinity ofseawater) for 15 days, prior to transfer to freshwater. “Adapted” asused herein, refers to a successful transition to the altered aquaticenvironment. After maintenance in water having decreasing salinity, ascompared to seawater, the marine fish are then placed into freshwaterhaving a PVCR modulator, as described herein. The fish can remain andgrow in freshwater, modified by the addition of PVCR modulators,indefinitely, so long as there is modulated or maintained expressionand/or sensitivity of the PVCR (e.g., maintained in modified freshwaterand fed an NaCl diet).

The invention further includes adding feed to the freshwater. Thefrequency and amount of feed that fish are fed, are taught in the art.Generally, the fish are fed 1-3 times a day, totaling about 0.25-0.5%body weight/day. The feed has enough NaCl to contribute to a modulatedor maintained level of the PVCR modulator in the serum of the marinefish. Specifically, the presence of sufficient amounts of NaCl in thefeed causes the marine fish to drink more water from the surroundingenvironment. Although NaCl decreases PVCR sensitivity, the ingestion offreshwater having at least one PVCR modulator causes an overall rise inthe serum level of PVCR modulator. The increase in serum levels of PVCRmodulator results in a modulation in expression of PVCR.

In another embodiment, the present invention is directed toward anaquatic mixture for providing an environment to transfer marine fish tofreshwater, comprising at least one PVCR modulator. An “aquatic mixture”is defined herein to mean a composition that provides a suitableenvironment for the successful transfer of marine fish to freshwater bythe methods of the present invention. The aquatic mixture can bepremixed for immediate use in the methods of the present invention.Alternatively, the aquatic mixture can require reconstitution withwater. The aquatic mixture when reconstituted yields a solutioncomprising about 0.3-10 mM Ca²⁺ and about 0.5-10 mM Mg²⁺. The aquaticmixture can optionally include an amino acid in an amount between about1 gm/kg and about 10 gm/kg.

The present invention also relates to an aquatic food composition. An“aquatic food composition” refers to fish feed, as described herein. Anaquatic food composition or feed suitable for use in the presentinvention contains between about 1%-10% of NaCl by weight, or betweenabout 10,000 mg NaCl/kg of feed and about 100,000 mg NaCl/kg of feed(e.g., 12,000 mg/kg). The feed is referred to herein as a “NaCl diet.”The NaCl can be combined with other sodium salts to confer the desiredeffect of modulating or maintaining PVCR expression, altering PVCRsensitivity and/or inducing the fish to drink more. Hence, as usedherein, the term NaCl, includes a substantially pure compound, mixturesof NaCl with other sources of sodium, or other sources of sodium. Thefeed can further include a PVCR modulator, and in particular a PVCRagonist such as an amino acid. In one embodiment, the feed has betweenabout 1% and about 10% NaCl by weight and an amino acid such astryptophan in an amount between about 1 and about 10 gm/kg. Thisembodiment is referred to herein as “APS Process II,” which is furtherdefined in Example 2.

The feed can be made in a number of ways, so long as the properconcentration of NaCl is present. The feed can be made, for example, byreformulating the feed, or by allowing the feed to absorb a solutionhaving the NaCl and optionally, adding a PVCR modulator. Additionally, atop dressing can be added for palatability. Example 3 describes indetail one way to make the feed. Alternate methods of preparing fishfeed are know to those of skill in the relevant art.

Another embodiment of the present invention includes feeding marine fishfeed having between 1% and 10% NaCl by weight when the fish aremaintained in a freshwater environment having between about 0.3 andabout 10.0 mM of calcium, and between about 0.5 mM and about 10.0 mM ofmagnesium. When this embodiment of the present invention is carried out,the levels of calcium, magnesium and/or sodium in the serum of themarine fish is increased, as compared to PVCR expression and/orsensitivity seen in freshwater fish.

In another embodiment, the fish, while in water having decreasesalinity, as compared to seawater, or while in the freshwater having thePVCR modulator, are also exposed to a photoperiod. A photoperiod refersto exposing the fish to light (e.g., sunlight, incandescent light orfluorescent light). Preferably, the photoperiod is substantiallycontinuous, or occurs long enough to increase growth. The photoperiodcan occur for at least about 12 hours within a 24 hour interval, or forlonger periods such as about 14, 16, 18, 20, 22 or preferably, about 24hours.

The methods of the present invention modulate or maintain the expressionand/or sensitivity of the PVCR in marine fish which results in reducedosmotic stress and in reduced mortality. Marine fish cultured infreshwater by methods of the present invention consume feed and exhibitgrowth. In contrast, marine fish that are not cultured in freshwater bymethods of the present invention experience osmotic stress, reduced orno food consumption, and eventually death. The osmotic stress resultsfrom differences in the osmotic pressure between the surroundingenvironment and body compartments of the fish. This disturbs thehomeostatic equilibrium of the fish and results in decreased growth,reproductive failure and reduced resistance to disease. The fish thathave undergone the steps of the present invention do not experience asignificant amount of osmotic stress. As a result, the fish are able togrow. Surprisingly, as described and exemplified herein, marine fishadapted by the present invention grow almost as well as marine fishmaintained in seawater (e.g., 53% increased growth in fish subjected tothe present invention for 37 days, as compared to 60% increased growthof fish maintained in seawater for 37 days). Additionally, marine fishcultured in freshwater by methods of the present invention exhibit asurvival rate that is significantly greater than the rate for marinefish that are transferred directly to freshwater and not subjected tothe steps of the present invention (e.g., between about 60% and about100%). See FIGS. 15 and 16.

The methods of the present invention also decrease the incidence ofdisease among the marine fish transferred to freshwater. Because thefish treated with the methods of the present invention experience lessstress upon transfer to freshwater, their immune functions are stronger,and they are less susceptible to parasitic, viral, bacterial and fungaldiseases. Thus, marine fish cultured by methods of the present inventionare healthier.

Methods Assessment of the PVCR

The present invention includes methods of detecting the level of thePVCR to determine whether fish are ready for transfer from seawater tofreshwater. Methods that measure PVCR levels include several suitableassays. Suitable assays encompass immunological methods, such as FACSanalysis, radioimmunoassay, flow cytometry, enzyme-linked immunosorbentassays (ELISA) and chemiluminescence assays. Any method known now ordeveloped later can be used for measuring PVCR expression.

Antibodies reactive with the PVCR or portions thereof can be used. In apreferred embodiment, the antibodies specifically bind with the PVCR ora portion thereof. The antibodies can be polyclonal or monoclonal, andthe term antibody is intended to encompass polyclonal and monoclonalantibodies, and functional fragments thereof. The terms polyclonal andmonoclonal refer to the degree of homogeneity of an antibodypreparation, and are not intended to be limited to particular methods ofproduction.

In several of the preferred embodiments, immunological techniques detectPVCR levels by means of an anti-PVCR antibody (i.e., one or moreantibodies). The term “anti-PVCR” antibody includes monoclonal and/orpolyclonal antibodies, and mixtures thereof.

Anti-PVCR antibodies can be raised against appropriate immunogens, suchas isolated and/or recombinant PVCR or portion thereof (includingsynthetic molecules, such as synthetic peptides). In one embodiment,antibodies are raised against an isolated and/or recombinant PVCR orportion thereof (e.g., a peptide) or against a host cell which expressesrecombinant PVC R. In addition, cells expressing recombinant PVCR, suchas transfected cells, can be used as immunogens or in a screen forantibody which binds receptor.

Any suitable technique can prepare the immunizing antigen and producepolyclonal or monoclonal antibodies. The art contains a variety of thesemethods (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur.J. Immunol. 6: 511-519 (1976); Milstein et al., Nature, 266: 550-552(1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D.Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.); Current Protocols In MolecularBiology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al.,Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Generally, fusing antibody producing cells with a suitable immortal ormyeloma cell line, such as SP2/0, can produce a hybridoma. For example,animals immunized with the antigen of interest provide the antibodyproducing cell, preferably cells from the spleen or lymph nodes.Selective culture conditions isolate antibody producing hybridoma cellswhile limiting dilution techniques produce them. Researchers can usesuitable assays such as ELISA to select antibody producing cells withthe desired specificity.

Other suitable methods can produce or isolate antibodies of therequisite specificity. Examples of other methods include selectingrecombinant antibody from a library or relying upon immunization ofanimals such as mice.

According to the method, an assay can determine the level of PVCR in abiological sample. In determining the amounts of PVCR, an assay includescombining the sample to be tested with an antibody having specificityfor the PVCR, under conditions suitable for formation of a complexbetween antibody and the PVCR, and detecting or measuring (directly orindirectly) the formation of a complex. The sample can be obtaineddirectly or indirectly, and can be prepared by a method suitable for theparticular sample and assay format selected.

In particular, tissue samples, e.g., gill tissue samples, can be takenfrom fish after they are anaesthetized with MS-222. The tissue samplesare fixed by immersion in 2% paraformaldehyde in appropriate Ringerssolution corresponding to the osmolality of the fish, washed in Ringers,then frozen in an embedding compound, e.g., O.C.T.™ (Miles, Inc.,Elkahart, Ind., USA) using methylbutane cooled with liquid nitrogen.After cutting 8-10μ tissue sections with a cryostat, individual sectionsare subjected to various staining protocols. For example, sectionsare: 1) blocked with goat serum or serum obtained from the same speciesof fish, 2) incubated with rabbit anti-CaR or anti-PVCR antiserum, and3) washed and incubated with peroxidase-conjugated affinity-purifiedgoat antirabbit antiserum. The locations of the boundperoxidase-conjugated goat antirabbit antiserum are then visualized bydevelopment of a rose-colored aminoethylcarbazole reaction product.Individual sections are mounted, viewed and photographed by standardlight microscopy techniques. One anti-CaR antiserum used to detect fishPVCR protein is raised in rabbits using a 23-mer peptide correspondingto amino acids numbers 214-236 localized in the extracellular domain ofthe RaKCaR protein (Riccardi et al., P.N.A.S. 92: 131-135 (1995);accession number NP 058692). The sequence of the 23-mer peptide is:ADDDYGRPGIEKFREEAEERDIC (SEQ ID NO.: 24) A small peptide with thesequence DDYGRPGIEKFREEAEERDICI (SEQ ID NO.: 25) or ARSRNSADGRSGDDLPC(SEQ ID NO.: 26) can also be used to make antisera containing antibodiesto PVCRs. Such antibodies can be monoclonal, polyclonal or chimeric.

Suitable labels can be detected directly, such as radioactive,fluorescent or chemiluminescent labels. They can also be indirectlydetected using labels such as enzyme labels and other antigenic orspecific binding partners like biotin. Examples of such labels includefluorescent labels such as fluorescein, rhodamine, chemiluminescentlabels such as luciferase, radioisotope labels such as ³²P, ¹²⁵I, ¹³¹I,enzyme labels such as horseradish peroxidase, and alkaline phosphatase,β-galactosidase, biotin, avidin, spin labels and the like. The detectionof antibodies in a complex can also be done immunologically with asecond antibody which is then detected (e.g., by means of a label).Conventional methods or other suitable methods can directly orindirectly label an antibody.

In performing the method, the levels of the PVCR are distinct from thecontrol. Varied levels or the presence of PVCR expression, as comparedto a control, indicate that the fish or the population of fish fromwhich a statistically significant amount of fish were tested, are readyfor transfer to freshwater. A control refers to a level of PVCR, if any,from a fish that is not subjected to the steps of the present invention,e.g., not subjected to freshwater having a PVCR modulator and/or not feda NaCl diet.

The PVCRs can also be assayed by Northern blot analysis of mRNA fromtissue samples. Northern blot analysis from various shark tissues hasrevealed that the highest degree of PVCRs expression is in gill tissue,followed by the kidney and the rectal gland. There appear to be at leastthree distinct mRNA species of about 7 kb, 4.2 kb and 2.6 kb. Forexample, the PVCRs can also be assayed by hybridization, e.g., byhybridizing one of the PVCR sequences provided herein (e.g., SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23), its complement or anoligonucleotide derived from one of the sequences, to a mRNA purifiedfrom tissue sample from a fish. Such a hybridization sequence can have adetectable label, e.g., radioactive, fluorescent, etc., attached, toallow the detection of hybridization product. Methods for hybridizationare well known, and such methods are provided in U.S. Pat. No.5,837,490, by Jacobs et al., the entire teachings of which are hereinincorporated by reference in their entirety. The design of theoligonucleotide probe should preferably follow these parameters: (a) itshould be designed to an area of the sequence which has the fewestambiguous bases (“N's”), if any, and (b) it should be designed to have aT_(m) of approx. 80° C. (assuming 2° C. for each A or T and 4 degreesfor each G or C).

Stringency conditions for hybridization refers to conditions oftemperature and buffer composition which permit hybridization of a firstnucleic acid sequence to a second nucleic acid sequence, wherein theconditions determine the degree of identity between those sequenceswhich hybridize to each other. Therefore, “high stringency conditions”are those conditions wherein only nucleic acid sequences which are verysimilar to each other will hybridize. The sequences can be less similarto each other if they hybridize under moderate stringency conditions.Still less similarity is needed for two sequences to hybridize under lowstringency conditions. By varying the hybridization conditions from astringency level at which no hybridization occurs, to a level at whichhybridization is first observed, conditions can be determined at which agiven sequence will hybridize to those sequences that are most similarto it. The precise conditions determining the stringency of a particularhybridization include not only the ionic strength, temperature, and theconcentration of destabilizing agents such as formamide, but also onfactors such as the length of the nucleic acid sequences, their basecomposition, the percent of mismatched base pairs between the twosequences, and the frequency of occurrence of subsets of the sequences(e.g., small stretches of repeats) within other non-identical sequences.Washing is the step in which conditions are set so as to determine aminimum level of similarity between the sequences hybridizing with eachother. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between two sequences results in a1° C. decrease in the melting temperature (T_(m)) for any chosen SSCconcentration. Generally, a doubling of the concentration of SSC resultsin an increase in the T_(m) of about 17° C. Using these guidelines, thewashing temperature can be determined empirically, depending on thelevel of mismatch sought. Hybridization and wash conditions areexplained in Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) onpages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.

High stringency conditions can employ hybridization at either (1) 1×SSC(10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calfthymus DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with high stringency washes of either (1) 0.3-0.1×SSC, 0.1% SDSat 65° C., or (2) 1 mM Na₂EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS at 65° C.The above conditions are intended to be used for DNA-DNA hybrids of 50base pairs or longer. Where the hybrid is believed to be less than 18base pairs in length, the hybridization and wash temperatures should be5-10° C. below that of the calculated T_(m) of the hybrid, where T_(m)in ° C.=(2× the number of A and T bases)+(4× the number of G and Cbases). For hybrids believed to be about 18 to about 49 base pairs inlength, the T_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

Moderate stringency conditions can employ hybridization at either (1)4×SSC, (10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (2) 4×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with moderate stringency washes of 1×SSC, 0.1% SDS at 65° C. Theabove conditions are intended to be used for DNA-DNA hybrids of 50 basepairs or longer. Where the hybrid is believed to be less than 18 basepairs in length, the hybridization and wash temperatures should be 5-10°C. below that of the calculated T_(m) of the hybrid, where T_(m) in °C.=(2× the number of A and T bases)+(4× the number of G and C bases).For hybrids believed to be about 18 to about 49 base pairs in length,the T_(m) in ° C.=(81.5° C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na⁺), and “L” is the length of the hybrid in base pairs.

Low stringency conditions can employ hybridization at either (1) 4×SSC,(10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H₂O (88 g/liter), pH to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calfthymus DNA at 50° C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 40° C., (3) 1% bovine serum albumen(fraction V), 1 mM Na₂.EDTA, 0.5 M NaHPO₄ (pH 7.2) (1 M NaHPO₄=134 gNa₂HPO₄.7H₂O, 4 ml 85% H₃PO₄ per liter), 7% SDS, 0.1-2 mg/ml denaturedcalf thymus DNA at 50° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 40° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 50° C., or (6) 5×SSC, 5× Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at40° C., with low stringency washes of either 2×SSC, 0.1% SDS at 50° C.,or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na₂EDTA, 40 mMNaHPO₄ (pH 7.2), 5% SDS. The above conditions are intended to be usedfor DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid isbelieved to be less than 18 base pairs in length, the hybridization andwash temperatures should be 5-10° C. below that of the calculated T_(m)of the hybrid, where T_(m) in ° C.=(2× the number of A and T bases)+(4×the number of G and C bases). For hybrids believed to be about 18 toabout 49 base pairs in length, the T_(m) in ° C.=(81.5°C.+16.6(log₁₀M)+0.41(% G+C)−0.61 (% formamide)−500/L), where “M” is themolarity of monovalent cations (e.g., Na⁺), and “L” is the length of thehybrid in base pairs.

Hence, the present invention includes kits for the detection of the PVCRor the quantification of the PVCR having either antibodies specific forthe PVCR protein or a portion thereof, or a nucleic acid sequence thatcan hybridize to the nucleic acid of the PVCR.

Alterations in the expression or sensitivity of PVCRs could also beaccomplished by introduction of a suitable transgene. Suitabletransgenes would include either the PVCR gene itself or modifier genesthat would directly or indirectly influence PVCR gene expression.Methods for successful introduction, selection and expression of thetransgene in fish oocytes, embryos and adults are described in Chen, TTet al., Transgenic Fish, Trends in Biotechnology 8:209-215 (1990).

The present invention is further and more specifically illustrated bythe following Examples, which are not intended to be limiting in anyway.

EXEMPLIFICATION Example 1 Polyvalent Cation-Sensing Receptors (PVCRs)Serve as Salinity Sensors in Fish

Polyvalent cation-sensing receptors (PVCRs) serve as salinity sensors infish. These receptors are localized to the apical membranes of variouscells within the fish's body (e.g., in the gills, intestine, kidney)that are known to be responsible for osmoregulation. A full-lengthcation receptor (CaR) from the dogfish shark has been expressed in humanHEK cells. This receptor was shown to respond to alterations in ioniccompositions of NaCl, Ca²⁺ and Mg²⁺ in extracellular fluid bathing theHEK cells. The ionic concentrations responded to encompassed the rangewhich includes the transition from freshwater to seawater. Expression ofPVCR mRNA is also modulated in fish after their transfer from freshwaterto seawater, and is modulated by PVCR agonists.

Using nucleic acid amplification with degenerate primers, partialgenomic clones of PVCRs have also been isolated from other fish species,including Cod (FIGS. 1A-B), Haddock (FIGS. 2A-B), Hake (FIGS. 3A-B),Halibut (FIGS. 4A-B), Mackerel (FIGS. 5A-B), Pollock (FIGS. 6A-B), SeaBass (FIGS. 7A-B), Swordfish (FIGS. 8A-B), Tuna (FIGS. 9A-B), WinterFlounder (FIGS. 10A-10C) and Summer Flounder (FIG. 11). The degenerateoligonucleotide primers used for isolating these clones, except forWinter Flounder, were 5′-TGT CKT GGA CGG AGC CCT TYG GRA TCG C-3′ (SEQID NO: 27) and 5′-GGC KGG RAT GAA RGA KAT CCA RAC RAT GAA G-3′ (SEQ IDNO: 28), where K is T or G, Y is C or T, and R is A or G. The degenerateoligos were generated by standard methodologies (Preston, G. M., 1993,“Polymerase chain reaction with degenerate oligonucleotide primers toclone gene family members,” in: Methods in Mol. Biol., vol. 58, ed. A.Harwood, Humana Press, pp. 303-312). Nucleic acids from these specieswere amplified, purified by agarose gel electrophoresis, ligated into anappropriate plasmid vector (Novagen's pT7 Blue or Promega's pGEM-T) andtransformed into an appropriate bacterial host strain (Novagens' NovaBlue Competent Cells or Promega's JM 109 competent cells). The plasmidsand inserts were purified from the host cells, and sequenced. FIGS.13A-C shows the deduced amino acid sequences and alignment for the PVCRsfrom Cod, Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass,Swordfish, Tuna and Winter Flounder.

A winter flounder lambda ZAP cDNA library was manufactured usingstandard commercially available reagents with cDNA synthesized from polyA+ RNA isolated from winter flounder urinary bladder tissue as describedand published in Siner et al. Am. J. Physiol. 270:C372-C381, 1996. Thewinter flounder urinary bladder cDNA library was plated and resultingphage plaques screened using a ³² P-labeled shark kidney calciumreceptor cDNA probe under intermediate stringency conditions (0.5×SSC,0.1% SDS, 50° C.). Individual positive plaques were identified byautoradiography, isolated and rescued using phagemid infections totransfer cDNA to KS Bluescript vector. The nucleotide (nt) sequence,FIG. 10A, (SEQ ID NO: 19) of the winter flounder PVCR clone was obtainedusing commercially available automated sequencing service that performsnucleotide sequencing using the dideoxy chain termination technique. Thededuced amino acid sequence (SEQ ID NO: 20) is shown in FIGS. 10B and10C. The winter flounder PVCR nucleotide sequence was compared to othersaquatic PVCR using commercially available nucleotide and proteindatabase services including GENBANK and SWISS PIR.

Example 2 Growth of Marine Fish in Freshwater Using the Methods of thePresent Invention

Methods:

The following examples refer to APS Process I and APS Process IIthroughout. APS stands for “AquaBio Products Sciences®, L.L.C.” APSProcess I is also referred to herein as “SUPERSMOLT™ I Process” or“Process I.” An “APS Process I” fish or smolt refers to a fish or smoltthat has undergone the steps of APS Process I. An APS Process I smolt isalso referred to as a “SUPERSMOLT™ I” or a “Process I” smolt. Likewise,APS Process II is also referred to herein as “SUPERSMOLT™ II Process” or“Process II.” An “APS Process II” fish or smolt refers to a fish orsmolt that has undergone the steps of APS Process II. An APS Process IIsmolt is also referred to as a “SUPERSMOLT™ II” or a “Process II” smolt.

APS Process I: Marine fish are exposed to or maintained in freshwatercontaining 0.3-10.0 mM calcium and 0.5-10.0 mM magnesium ions. Thiswater is prepared by addition of calcium carbonate and/or calciumchloride and magnesium chloride to the freshwater. Fish are fed withfeed pellets containing 1-7% (weight/weight) NaCl. See Example 3 forfurther details regarding the feed. Fish are exposed to or maintained inthis regimen of water mixture and feed for a total of 30-45 days, usingstandard hatchery care techniques. Water temperatures vary between10-16° C. Fish are exposed to a constant photoperiod for the duration ofAPS Process I. A fluorescent light is used for the photoperiod.

APS Process II: Marine fish are exposed to or maintained in freshwatercontaining 0.3-10.0 mM calcium and 0.5-10.0 mM magnesium ions. Thiswater is prepared by addition of calcium carbonate and/or calciumchloride and magnesium chloride to the freshwater. Fish are fed withfeed pellets containing 1-7% (weight/weight) NaCl and either 2 gm or 4gm of L-Tryptophan per kg of feed. See Example 3 for further detailsregarding the feed. Fish are exposed to or maintained in this regimen ofwater mixture and feed for a total of 30-45 days using standard hatcherycare techniques. Water temperatures vary between 10-16° C. Fish areexposed to a constant photoperiod for the duration of APS Process II. Afluorescent light is used for the photoperiod.

Summer Flounders of various weights that were all derived from a singlehomogenous stock of farm raised animals (Great Bay AquaFarms Portsmouth,N.H.) were transported and placed in artificial seawater (Crystal Sea)within the APS laboratory. These were divided into two groups (n=13) andone maintain in seawater (Seawater Control) for a total of 81 days andfed a standard flounder diet (Corey Feeds, New Brunswick, Canada). Theother (Freshwater) was adapted to APS Process I conditions over 30 daysconsisting of 5 mM Ca²⁺, 8 mM Mg²⁺ concentrations in the water and a1.2% NaCl supplemented diet of 70% standard flounder feed (Corey Feeds,New Brunswick, Canada) and 30% ground squid. These flounder were thenmaintained in APS Process I conditions for a total of 51 days and theirgrowth compared to that exhibited by matched paired summer floundermaintain in seawater.

Flounders were adapted to the APS Process I by the following 30 dayschedule:

-   1. Maintenance in seawater for 5 days.-   2. Reduce water salinity to 50% seawater for 10 days.-   3. Reduce water salinity to 25% seawater for 15 days.-   4. Place fish in APS Process I water (5 mM Ca²⁺, 8 mM Mg²⁺    concentrations in the freshwater, pH 7.6-8.0)

Fish were individually tagged using colored elastomer tags their changein weight was determined at specific time points during the 51 dayexperimental interval.

The feed conversion ratio or FCR is obtained by dividing the body weightgained by a group of fish into the amount of food fed to the group offish. The more efficient the conversion of food into body weight growthby fish, the smaller the FCR (small amount of food/large weight gain offish). A very small FCR number (less than 1) encompasses a highlyefficient conversion of food into body weight growth which is the goalof aquaculture. By contrast, a large FCR means an inefficient conversionof food into body weight growth and is generally undesirable. A large orpoor FCR is undesirable due to the cost of feed and the necessity to usemore feed to grow fish to a given weight. The FCR values for fishsubjected to the methods of the present invention are generally smallerand more desirable, in some instances (e.g., when fish were fed dryfeed), than most industry published values because the present inventioneliminates the presence of osmotically damaged fish that tend toincrease the overall FCR since they eat food but do not grow. Themethods of the present invention, result in a lower FCR, allowingoptimal feeding and growth of most fish. The FCR of fish subjected tothe present invention is sufficient to maintain growth and feeding ofthe majority of fish, or preferably increase the growth and feedconsumption of the majority of fish. When fish are subjected to themethods of the present invention, they exhibit ranges of FCRs, forexample, would include values between about 0.7 and about 7.0. Inparticular, food consumption or food intake is improved because it isbelieved that the fish “smell” or “sense” the food with the PVCR incells of the olfactory lamellae or olfactory bulb.

The specific growth rate (SGR) of the fish was determined by dividingthe weight of the fish at the end of the given time point by thestarting weight of the fish.

All calculations to obtain feed conversion ratio (FCR) or specificgrowth rate (SGR) and growth factor (GF3) were performed using standardaccepted formulae (Willoughby, S. Manual of Salmonid Farming BlackwellScientific, Oxford UK 1999).

Results and Discussion:

A marine fish, Summer Flounder, can be adapted and grown under APSProcess I conditions for a prolonged interval (51 days) with growthrates similar to that exhibited by matched control Summer Flounder inseawater.

Tables I and II display data obtained from identical groups of summerflounder maintained under either seawater (seawater control) or APSProcess I freshwater conditions. Water quality and temperatures (16.3°C. vs 17.9° C. average) were comparable. Flounders were successfullyadapted to APS Process I conditions without significant mortalities andtheir overall appearance did not differ significantly from those matchedcontrols that were maintained in seawater. TABLE I Growth of SummerFlounder in Freshwater. APS Freshwater Total Weight Weight Weight WeightWeight Flounder # Start 20 days 37 days 51 days Gained 116 1 161 145 144140 −21 118 2 87 94 123 3 60 142 4 94 104 115 112 18 146 5 73 73 221 6118 135 145 156 38 223 7 105 225 8 96 112 124 133 37 226 9 156 183 203221 65 227 10 162 176 172 180 18 233 11 205 207 220 244 39 234 12 221224 235 13 150 161 164 174 24 Average 129.8462 146.7273 160.875 170 27.2S. Dev. 50.15 48.34065 36.65452 44.75648 p test 0.017186 0.013243 0.0085Amount Fed (gm) 342 315 291 948 water ° C. 17.9 19.4 16.4 17.9 (Average)FCR 3.96 SGR 0.53% bw/dayFeed conversion ratio (FCR);specific growth rate (SGR)

TABLE II Growth of Summer Flounder in Seawater. APS Seawater Total days51 Weight Weight Weight Weight Total Weight Fish # Start 20 days 37 days51 days Gained 117 114 118 147 146 168 168 21 120 60 70 91 94 34 122 90115 142 153 63 126 128 142 174 196 68 127 67 76 93 105 38 130 95 90 9386 −9 131 92 87 101 104 12 132 93 101 121 127 34 134 174 191 235 236 62139 116 140 121 138 170 175 54 145 79 87 100 135 56 Average 105.8462 113135.2727 143.5455 39.36364 S. Dev. 31.997 37.26392 46.82327 47.05181 Ttest 0.308845 0.041076 0.014804 Amount 301 324 254 879 Fed (gm) FCR 1.99SGR 0.6% bw/daybody weight = bw

Overall mortalities of fish during the 51 day test interval was lower inseawater ({fraction (2/13)} or 15.4%) as compared to floundersmaintained under APS Process I conditions ({fraction (5/13)} or 38.5%).The average weight gained by all flounders maintain under APS Process Iconditions (27.2 gm) was less as compared to overall weight gain of theseawater control group (38.4 gm). Significant weight gains were observedin both groups after intervals of 20 days for APS Process I fish and 37days for flounder maintained in seawater. Thus, the average specificgrowth rates (SGR) amongst the surviving flounders in APS Process I(0.53% body weight per day) were comparable to those maintained inseawater (0.6% body weight per day).

In contrast, 100% of marine fish (Cod, Haddock, Hake, Halibut, Mackerel,Pollock, Sea Bass, Swordfish, Tuna, Winter Flounder and Summer Flounder)die within 72 hours of freshwater transfer.

Comparison of the food conversion ratio (FCR) between floundersmaintained in APS Process I vs seawater shows that flounders maintainedunder APS Process I conditions displayed a significantly greater FCR(3.96), as compared to their matched seawater controls (1.99).

FIGS. 1A-B show the individual weight gain performances of taggedflounders maintained under APS Process I or seawater conditions. It isnotable that there are wide variations in individual growth rates suchthat some flounders (e.g. #9 and #11) exhibited steady and significantgrowth under APS Process I conditions while others showed poor weightgains (e.g. #10) or even lost weight (e.g. #1). Similar performancecharacteristics were observed for flounder in seawater although thevariation in individual performances were less pronounced as compared toflounders maintained in APS Process I.

Taken together, these data demonstrate that summer flounder can besuccessfully maintained under freshwater conditions using APS Process Ifor a prolonged interval (51 days) of time. Under normal conditions,summer flounder growth and survival are normally restricted toapproximately 25% seawater whereupon the flounders die if the salinityis further reduced. These data form the basis of culture of summerflounder in freshwater environments distant from the marine environmentitself where prices for flounder fillets would more than offset thepoorer performance (increased mortalities and poorer FCR and weightgains) as compared to seawater controls.

Transferring marine fish to freshwater using APS Process II is expectedto provide even better growth rates, than seen with APS Process I.Salmon and Trout that underwent APS Process II exhibited significantincreases in growth rates, as illustrated in related applications,patent application Ser. Nos. 09/687,372; 09/687,476; 09/687,477, allentitled, “Methods for Raising Pre-Adult Anadromous Fish,” and patentapplication Ser. No. 09/687,373, entitled “Growing Marine Fish in FreshWater”, all filed on Oct. 12, 2000.

Example 3 The Feed

Two general methods were used to prepare feed for consumption by fish aspart of APS Process I and II. These two processes involve eitherreformulation of feed or addition of a concentration solution forabsorption by the feed followed by a top dressing for palatability. Thisdisclosure describes the methodology to prepare feed using each of these2 methods.

Methods:

Feed Manufacture for Salmon Experiments

To reformulate feed, the ingredients are as follows: Base Diet was madeusing the following ingredients and procedure: 30% Squid (liquefied inblender), 70% Corey Aquafeeds flounder diet (powderized in blender).Ingredients were blended into a semi moist “dough” ball. Otheringredients including NaCl or PVCR active compounds were blended intothe base diet by weight according to experimental parameters.

Moore Clark standard freshwater salmonid diet (sizes 1.2, 1.5, 2.0, 2.5,and 3.5 mm) can also be used. A top dressing was applied to the pelletssuch that top dressing is composed of 4% of the weight of the Base Diet.Top dressing is composed of 50% krill hydrolysate (Specialty MarineProducts Ltd.) and 50% Menhaden fish oil. The top dressing is added forpalatability and sealing of added ingredients.

Other ingredients can include NaCl, MgCl₂, CaCl₂ or L-Tryptophan thatare added by weight to the base diet by weight, as described herein.

Preparation of Feed Containing 7% (Weight/Weight) NaCl:

For the APS Process I: Solid NaCl or NaCl apportioned at a ratio of 7%of the weight of the Moore Clark standard freshwater salmonid dietweight was added to a volume of tap water approximately 3-4 times theweight of NaCl. The mixture was heated to 60-70° C. with mixing via useof a magnetic stirring bar to dissolve salt. The NaCl solution was thenpoured into a hand held sprayer and applied to the Moore Clark standardfreshwater salmonid diet that is tumbling inside of a 1.5 cubic metermotorized cement mixer. After absorption of the NaCl rich solution, thewetted Moore Clark standard freshwater salmonid diet is spread outthinly on window screening and placed in an enclosed rack systemequipped with a fan and 1500 watt heater to expedite drying process.After drying for approximately 6 hr, the dried NaCl-rich pellets arereturned to the cement mixer and a top dressing is applied. The feed isstored at room temperature until use.

Preparation of Feed Containing 7% (weight/weight) NaCl+PVCR Agonist(Tryptophan) For the APS Process II: Solid sodium chloride or NaClapportioned at a ratio of 7% of the weight of the Moore Clark standardfreshwater salmonid diet weight was added to a volume of tap waterapproximately 3-4 times the weight of NaCl. The mixture was heated to60-70° C. with mixing via use of a magnetic stirring bar to dissolvesalt. USP Grade L-Tryptophan was added to the water at either 2 grams or4 grams for every kg of Moore Clark standard freshwater salmonid dietdepending on formulation need. Dilute hydrochloric acid was added to thewater with mixing until the tryptophan was dissolved and the pH ofsolution was approximately 4.0. The NaCl+tryptophan solution was thenpoured into a hand held sprayer and was then applied to the Moore Clarkstandard freshwater salmonid diet tumbling inside a cement mixer. Afterabsorption of the NaCl+tryptophan solution, the wetted Moore Clarkstandard freshwater salmonid diet is then spread out thinly on windowscreening and placed in an enclosed rack system equipped with a fan and1500-watt heater to expedite drying process. After drying forapproximately 6 hr, the dried NaCl/tryptophan-rich pellets are thenreturned to the cement mixer and a top dressing is applied. The feed isstored at room temperature until use.

Example 4 DNA and Putative Protein Sequences from Partial Genomic Clonesof Polyvalent Cation Receptor Protein Amplified by PCR from the DNA ofSeveral Species of Marine Fish

These data provide the partial PVCR genomic sequences isolated in 13species of marine fish. Each of these nucleotide sequences is unique andthus could be used as a unique probe to isolate the full-length cDNAfrom each species. Moreover, these nucleotide sequences could form thebasis for a specific assay kit(s) for detection of PVCR expression invarious tissues of these fish. For example, the kit could optionallyinclude a labeled hybridization probe suitable for in situhybridization.

The PVCR has been isolated in several species including Cod, Haddock,Hake, Halibut, Mackerel, Pollock, Sea Bass, Swordfish, Tuna, WinterFlounder and Summer Flounder. Sequences of mammalian CaRs together withthe nucleotide sequence of SKCaR (FIGS. 14A and 14B) were used to designdegenerate oligonucleotide primers to highly conserved regions in theextracellular and transmembrane domains of polyvalent cation receptorproteins using standard methodologies (See GM Preston, “Polymerase chainreaction with degenerate oligonucleotide primers to clone gene familymembers,” Methods in Mol. Biol. Vol. 58, Edited by A. Harwood, HumanaPress, pages 303-312, 1993). Using these primers, cDNA or genomic DNAfrom various fish species representing important commercial products areamplified using standard PCR methodology. Amplified bands are thenpurified by agarose gel electrophoresis and ligated into appropriateplasmid vector that is transformed into a bacterial strain. After growthin liquid media, vectors and inserts are purified using standardtechniques, analyzed by restriction enzyme analysis and sequenced whereappropriate. Using this methodology, nucleotide sequences wereamplified.

To generate this sequence data, DNA was isolated from tissue samples ofeach of the species indicated using standard published techniques. DNAwas then amplified using polymerase chain reaction (PCR) methodologyincluding 2 degenerate PCR primers (DSK-F3 (5′-TGT CKT GGA CGG AGC CCTTYG GRA TCG C-3′; SEQ ID NO.: 29) and DSK-R4; (5′-GGC KGG RAT GAA RGAKAT CCA RAC RAT GAA G-3′ SEQ ID NO: 30). Amplified DNAs were thenpurified by agarose gel electrophoresis, subcloned into plasmid vectors,amplified, purified and sequenced using standard methods.

FIGS. 12A-C show an aligned genomic DNA sequences of 593 nucleotides for12 marine fish species, each of which codes for an identical region ofthe PVCR protein. Note that each nucleotide sequence derived from eachspecific species is unique. However, alterations in the DNA sequences ofthese genes often occur at common specific nucleotides within eachsequence of 593 nucleotides.

FIGS. 13A-C show aligned corresponding predicted protein sequencesderived from genomic nucleotide sequences displayed in FIGS. 12A-D. Notethat few alterations in the amino acid sequence of this portion of thePVCR occur as a consequence of alterations in the nucleotide sequence asshown in FIGS. 12A-D. All of these changes (e.g., Ala to Val; Arg toLys; and Cys to Tyr) are known as “conservative” substitutions of aminoacids in that they preserve some combination of the relative size,charge and hydrophobicity of the peptide sequence.

All cited references, patents, and patent applications are incorporatedherein by reference in their entirety. Also, companion patentapplication Ser. No. 10/268,051, entitled “Methods for Growing andImprinting Fish Using Odorant,” filed Oct. 11, 2001; patent applicationSer. No. 09/975,553, entitled “Methods for Raising Pre-adult AnadromousFish,” filed Oct. 11, 2001; International Application No.PCT/US01/31704, entitled “Polyvalent Cation-sensing Receptor Proteins inAquatic Species,” filed Oct. 11, 2001. Additionally, patent applicationSer. No. 09/687,477, entitled “Methods for Raising Marine Fish,” filedon Oct. 12, 2000; patent application Ser. No. 09/687,476, entitled“Methods for Raising Marine Fish,” filed on Oct. 12, 2000; patentapplication Ser. No. 09/687,373, entitled “Methods for Raising MarineFish,” filed on Oct. 12, 2000; Provisional Patent Application No.60/240,392, entitled “Polyvalent Cation Sensing Receptor Proteins inAquatic Species,” filed on Oct. 12, 2000; Provisional Patent ApplicationNo. 60/240,003, entitled “Polyvalent Cation Sensing Receptor Proteins inAquatic Species,” filed on Oct. 12, 2000, are all hereby incorporated byreference in their entirety. Additionally, application Ser. No.09/162,021, filed on Sep. 28, 1998, International PCT application No.PCT/US97/05031, filed on Mar. 27, 1997, and application Ser. No.08/622,738 filed Mar. 27, 1996, all entitled, “Polycation SensingReceptor in Aquatic Species and Methods of Use Thereof” are all herebyincorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes can be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1. A method of growing marine fish in freshwater, comprising: a) addingat least one Polyvalent Cation Sensing Receptor (PVCR) modulator tofreshwater in an amount sufficient to modulate or maintain expressionand/or sensitivity of at least one PVCR in one or more tissues; b)transferring the marine fish to the freshwater, modified according tostep a); and c) adding feed for fish consumption to the modifiedfreshwater, wherein the feed contains an amount of NaCl sufficient tocontribute to a significant increased level of said PVCR modulator inserum of the marine fish.